Echinops extract with anti-cancer activity

ABSTRACT

The present invention relates to an Extract of  Echinops spinosus  L (Asteraceae) and organic solvent soluble fractions of the extract that may be used in the treatment of cancer.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to an extract of Echinops spinosus L. (Asteraceae)and fractions thereof for use in the treatment of cancer; the inventionalso relates to the preparation of the extract and the fractions andpharmaceutical compositions containing them.

(b) Description of Prior Art

Cancers are uncontrolled cell proliferations that result from theaccumulation of genetic changes in cells endowed with proliferativepotential. After a variable latency period during which they areclinically silent, the malignant cells progress to aggressive invasiveand metastatic stages with tumor formation, bleeding, susceptibility toinfections, and wide-spread dissimination throughout the body.

Despite important advances in treatment, cancers still account for 28%of death in Western countries. Treatment of cancer has relied mainly onsurgery, chemotherapy, radiotherapy and more recently immunotherapy.Significant improvement in outcome has occurred with the use of combinedmodalities, for a small number of cancers. However, for the mostfrequent types of cancers (lung, breast, colo-rectal and the leukemias)complete remission and cure has not been achieved. Therefore, thedevelopment of new approaches for treating cancer patients is criticallyneeded particularly for those patients whose disease has progressed to ametastatic stage and are refractory to standard chemotherapy.

The leaves of Echinops spinosus L. (Asteraceae) have previously beenused in local or herbal medicine to treat warts; and a peptide havingvasoconstrictive properties has been isolated from the root of theplant.

It would be highly desirable to be provided with a novel therapy forcancer which overcome the drawbacks of the method of the prior art.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a dried extract of theroots of Echinops spinosus L. (Asteraceae) and fractions thereof thathave been found to be useful in treating cancers.

In accordance with one aspect of the invention there is provided ananti-cancer pharmaceutical composition comprising an acceptable,effective anti-cancer amount of an extract of roots Echinops spinosus L.(Asteraceae) or an organic solvent soluble fraction thereof, inassociation with a pharmaceutically acceptable carrier.

In accordance with another aspect of the invention there is provided anextract of roots of Echinops spinosus L. (Asteraceae) or an organicsolvent soluble fraction thereof, for use in the treatment of cancer.

In accordance with yet another aspect of the invention there is provideda method of treating cancer comprising administering to a patient, anacceptable, effective anti-cancer amount of an extract of roots ofEchinops spinosus L. (Asteraceae) or an organic solvent soluble fractionthereof.

In accordance with still another aspect of the invention there isprovided a method of producing an anti-cancer agent comprising organicsolvent extraction of an extract of roots of Echinops spinosus L.(Asteraceae) to produce an organic solvent soluble extract, andchromatographic separation of fractions of said organic solvent solubleextract.

For the purpose of the present invention the following terms are definedbelow.

The term “anti-cancer therapy” is intended to mean growthinhibition/eradication of primary tumors, stabilization of tumor growth,inhibition of metastasis formation, or prevention of tumor formation.Furthermore, anticancer activity also covers any combination between oursubstances and other known or investigational anticancer agents, inorder to improve the therapeutic efficacy of drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the in vitro cytotoxicity of various fractions usinghuman breast adenocarcinoma cell line, MCF7;

FIG. 1B illustrates the in vitro cytotoxicity of various fractions usinghuman ovarian adenocarcinoma cell line, A2780;

FIG. 2 illustrates the Lung Lewis carcinoma model schedule for drugtreatment and also the antimetastatic activity of 75B;

FIG 3A and 3B illustrate a schedule for drug treatment showing thatdoses used for the antimetastatic study have minor toxic effects to thehost, since toxicity was observed only with very high doses >300; and

FIG. 4 illustrates a schematic representation of the process offractionation.

DETAILED DESCRIPTION OF THE INVENTION

The organic solvent soluble fractions of the present invention arefractions soluble in organic solvents such as methanol, ethanol, ethylacetate and dimethyl sulfoxide.

The invention is more especially concerned with the fractions which aresoluble in the organic solvents but not soluble in water.

i) Extract of Echinops spinosus L. (Asteraceae)

ii) Fractions

The dried extract of Echinops spinosus L. (Asteraceae) is first treatedwith water to remove water soluble fractions of the extract, whereafterthe residue is treated with an organic solvent, for example, ethylacetate to dissolve the organic solvent soluble fractions.

The ethyl acetate soluble fractions are purified, for example, oncharcoal and/or CELITE™ (trademark for diatomaceous earth) andindividual fractions are separated by high-performance liquidchromatography.

The fractions recovered were dissolved in dimethyl sulfoxide or ethanol.

The samples when stored at −80° C. retained their activity even afterfour months.

The organic solvent soluble fractions and extract were found to have astrong antiproliferative activity in a panel of human cancer cell linesderived from breast, ovary, prostate and lung. In vivo, the fractionsand extract demonstrate antimetastatic activity in animal models.

The process of fractionation is illustrated schematically in FIG. 4.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

EXAMPLE I In Vitro Antiproliferative Activity

Cell Lines and Cell Culture

Two cell lines were used to test for the antiproliferative activity ofvarious fractions: human ovarian adenocarcinoma cell line A2780, and thehuman breast cancer cell line MCF7. Cells were grown in RPMI mediumsupplemented with 10% fetal bovine serum and penicillin-streptomycinantibiotics. Cells were maintained in culture at 37° C. in an atmosphereof 5% CO₂.

Cytotoxicity Assay

Exponentially growing cells (2−3×10³ cells/100 μl) were seeded in96-well plates and incubated for 16 h. Cells were then treatedcontinuously with the fractions. 72 h later, cell survival was evaluatedby replacing the culture media with 150 μl fresh medium. containing 10mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid buffer, pH 7.4and 50 μl of 2.5 mg/ml of3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) inPBS, pH 7.4, were then added. After 3-4 h of incubation at 37° C., themedium and MTT were removed, and 200 μl of DMSO (dimethyl sulfoxide) wasadded to dissolve the precipitate of reduced MTT, followed by additionof 25 ml glycine buffer (0.1M glycine plus 0.1M NaCl, pH 10.5). Theformazan crystals were then dissolved and the absorbance was determinedat 570 nm with a microplate reader (BIORAD, model 450). The MTT assaydistinguishes between viable and non-viable cells on the basis thatphysiologically active mitochondria metabolizes the MTT only in viablecells. The IC50 was calculated as the concentration of drug causing a50% inhibition in the absorbance compared to cells treated with solventalone.

Apoptosis Assay

Cells were seeded at 1×10⁶ cells/T75 cm² plate, then left to attachovernight. The cells were then continuously exposed to the extracts for72 hrs. Cells were then collected and washed 2× with PBS and thendiluted to 1×10⁶/100 μl PBS and placed in 96 well plate. Fixation wasperformed with 200 ml of 70% Ethanol with shaking at 4° C. for 30 min.Cells were then washed 1× with PBS, and permeabilized with 1% TRITON™X-100 in 0.1% sodium citrate on ice for 2 min. Cells were washed 2× withPBS, and then labeled in 50 ml/well TUNEL reaction mixture of theBoehringer Mannheim In Situ cell death detection kit at 37° C. in thedark for 1 hr. Cells were then washed 3× with 1% BSA in PBS andresuspended in 500 μl PBS for analysis by flow cytometry. The cell deathTunnel assay estimates the extent of DNA fragmentation. The fragmentedDNA is labeled at the free 3′ OH group using terminal deoxynucleotidetransferase. Fluoroscein labels are incorporated into nucleotidepolymers that are attached to the DNA fragments. The labeling isspecific to fragmented DNA and not degraded DNA due to the requiredpresence of the 3′ OH group. Thus, the level of fluorescence as measuredby a flow cytometer is correlated to the level of DNA fragmentation, andhence to the number of apoptotic cells.

Results

Methanol soluble fractions, but not water soluble fractions, was foundto have a potent antiproliferative activity in both A2780 and MCF7 celllines. Further chromatographic fractionation of these organic solubleextract led to the isolation of 12 fractions referred to as E1-E12(FIGS. 1A and 1B).

Morphological changes of cells treated with E fractions resembled thosedescribed with apoptosis-mediated cell death. Apoptosis was examinedusing ladder DNA and TUNEL Assays; it was found that some fractions,e.g. E4 and E5, induce apoptosis.

EXAMPLE II In Vivo Study

Lewis Lung Carcinoma Cell Line and Cell Culture

The Lewis lung carcinoma clone, M47, with a high metastatic potential tothe lung, was established and characterized (Brodt P., Cancer Res., 46:2442, 1986). These cells were confirmed to be free of mycoplasmainfection. Cells were maintained in RPMI-1640 medium supplemented with10% fetal bovine serum and 1% penicillin-streptomycin, under 5% CO₂.Cells were passaged twice a week. Stocks of cells were generated andstored as early passages (passage no. 8-10 considering the initial stockreceived as passage no. 1). Cells were then propagated and stocks of thesame passages were established and stored in liquid nitrogen for furtherstudies with AETERNA compounds.

For tumor induction, cells were grown to 70% confluence in completemedium and then collected using trypsin-EDTA solution [0.05% trypsin,0.53 mM EDTA-4Na in H_(B)SS without Ca++, Mg++, and NaHCO3; Cellgro no.25-052-Li]. Cells were then centrifuged and washed three times withphosphate buffer solution [D-PBS, Ca++ and Mg++ free; Cellgro no.21-031-LV], and resuspended at a dilution of 0.1 to 1×106 cells/0.1 ml.Viability was examined by trypan blue staining and only flasks in whichthe viability was >95% were used for in vivo studies.

The mouse strain used in this study is C57BL/10 from the researchlaboratories. The animal room used has two doors, one serving as theentrance, and the other door provides direct access towashing/sterilization/incineration accurate adjustment of environmentalparameters including temperature, humidity, ventilation, and lighting.Cleaning and sanitation practices are performed, on a daily basis, bypersonnel with appropriate training.

Tumor Cell Inoculation and Treatment

Animals were housed 5 per cage and were fed a diet of animal chow andwater ad libitum. After one week acclimatization, LLC cells weretransplanted subcutaneously, as a suspension of tumor cells [2−5×10⁵viable cells per 0.1 ml], in the axillary region of the right flank. Allanimals were inoculated at the same site. Animals were subjected, on adaily basis, to general examination. Tumor growth was monitored everysecond or third day using calipers. Parameters measured are: tumormeasured along the longest axis (length) and the perpendicular shortestaxis (width) and the relative tumor volume (in cm³) was calculated bythe formula: [Length (cm) ×(width cm)²]/2. When the tumor reaches a sizeof 0.5-1.0 cm² (approximately 2-3 weeks), mice were randomized intothree groups.

Animals were subjected to surgery to remove the primary tumor. The micewere lightly anesthetized with Forane. The skin overlying the tumor wascleaned with betadine and ethanol, in a laminar flow hood. A small skinincision (0.5-1 cm) was made using a sterile scalpel, and the tumor wascarefully separated from the normal tissues (skin and muscle). LLC (atearly stage of growth; 1-3 weeks) is well localized tumor and separationwas easy to achieve without any significant damage to normal tissues.The tumor was removed, weighed and fixed for histopathology purposes.The wound was closed with surgical stainless steel clips (Autoclips; 9mm; Clay Adams, Inc., Parsippany, N.J.). This site was furtherdisinfected with betadine and the animal was housed as described above.

Mice were randomized after surgery into a group of 5 per cage. Cageswere randomly assigned to specific experimental groups. The mice werethen labeled by numbers using the “ear punching” method. Mice werechecked on a daily basis to ensure the absence of infection. Animalswith discomfort were sacrificed immediately. An additional extra-groupof control mice was included to determine the optimal timing forsacrifice in order to obtain a significant number of well localized lungmetastases. This group was subjected to the same experimental procedureas group 1 with the exception of drug treatment. Based on this group, aperiod of two weeks after removal of the primary tumor was sufficient toobtain an average of 20-30 nodules on the lung surface. Therefore, a twoweek period after primary tumor removal was used to sacrifice treatedmice.

Dosing Schedule and Treatment

Drugs were given by gavage, using a 22 G curved feeding needle [totalvolume of 0.5 ml per animal], on a single daily basis administrationafter tumor cell inoculation. Control animals were given the same volumeof saline solution [0.9% sodium chloride; Abbott Lab., lot no. 12 455WS]. The dose of each drug was normalized to an average of 20 g bodyweight per animal. The schedules for drug treatment were based uponconditions described in FIGS. 2 and 3.

Animal Sacrifice, Tumor/organs Preparation

At the end of each experiment (a total of 5-8 weeks), animals weresacrificed in a CO₂ Chamber and autopsied. Tumors, organs or both wereremoved under sterile conditions [using a laminar flow hood]. Tumorswere weighed. Organs (5 per group) were examined for gross pathologicalchanges and then fixed in 10% formalin. Lungs were fixed in 10% Bouin'sfixative diluted in a formalin solution, and lung surface metastaseswere counted using a stereomicroscope at 4× magnification or amagnifying-glass, and then lungs were embedded in paraffin wax accordingto standard procedures. Embedded tissues were stored for futurehistopathological studies.

Statistical Analysis

The umpaired Student t-test was used to compare statistical significanceamong various groups.

Results

Methanol soluble fraction was found to have a good antimetastaticactivity in the Lewis lung carcinoma model. A dose relationship was alsoobserved (see FIGS. 2 and 3).

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A method of treating cancer comprisingadministering to a patient a therapeutically effective anti-canceramount of an extract of roots of Echinops spinosus L. (Asteraceae) or anorganic solvent soluble fraction thereof, in association with apharmaceutically acceptable carrier.